应用免疫磁珠捕获技术分析宿主细胞蛋白抗体覆盖率

Analysis of anti⁃host cell protein antibody coverage rate by the immunomagnetic beads separation technique

目的:建立并应用基于免疫磁珠捕获分离(immunomagnetic beads separation,IMBS)技术的宿主细胞蛋白(host cell protein,HCP)抗体覆盖率分析方法。方法:首先,建立和优化IMBS方法,包括HCP抗体磁珠偶联、洗涤和洗脱条件关键步骤。其次,针对二维电泳实验部分,设计等电聚焦质控和银染质控,并建立IMBS处理后的样品前处理方法,可用于LC⁃MS/MS分析,计算HCP抗体覆盖率。结果:优化确定IMBS覆盖率方法步骤,相对0.5 mg抗体,采用500μL磁珠偶联使HCP抗体的捕获效率最高,通过8次洗涤消除非特异性结合HCP,3次洗脱步骤充分收获结合的HCP。开发基于荧光标记多肽的2种等电聚焦质控品和银染灵敏度质控品,可以有效监控二维电泳实验过程。MS法具有较高的分辨率和测量精度,采用IMBS结合MS法可以识别到更多的HCPs,表现出来的覆盖率也高于IMBS结合二维电泳的方法;同时,IMBS结合MS法还可以通过识别高风险HCPs评估抗体的性能。结论:本研究建立稳定可靠的IMBS⁃2DE方法和IMBS⁃LC/MS/MS方法用于分析HCP抗体覆盖率,2个技术平台获得的抗体覆盖率水平由于分析灵敏度的不同导致结果有较大的差异。

Objective:To establish an immunomagnetic beads separation(IMBS)method for the application in the analysis of host cell protein(HCP)antibody coverage.Methods:Firstly,the IMBS method was developed and optimized,including key steps such as HCP antibody magnetic bead coupling,washing,and elution conditions.Secondly,for the two⁃dimensional electrophoresis experiments,isoelectric focusing and silver staining quality control materials were designed,and a sample pre⁃processing method was also established for IMBS⁃treated samples.The developed methods are applicable to LC⁃MS/MS to calculate the HCP antibody coverage.Results:The optimized IMBS method was developed and the highest capture efficiency of HCP antibody was achieved by coupling 500μL magnetic beads with 0.5 mg antibody.Eight washing steps were employed to eliminate nonspecific binding of HCP,and three elution steps were performed to thoroughly harvest bound HCP.Two isoelectric focusing quality control samples based on fluorescently labeled peptides and a silver staining sensitivity control sample were developed to effectively monitor the two⁃dimensional electrophoresis experiment process.The MS is of high resolution and measurement accuracy.When combining MS with IMBS,more HCPs can be identified than the combination of IMBS with two⁃dimensional electrophoresis.Moreover,the coverage demonstrated by the IMBS combined with mass spectrometry method is higher than that of IMBS combined with two⁃dimensional electrophoresis.Additionally,the IMBS combined with MS can assess antibody performance by recognizing high⁃risk HCPs.Conclusion:IMBS⁃2DE method and IMBS⁃LC/MS/MS method,which are stable and reliable,analyzing HCP antibody coverage were established,respectively.As a result,the antibody coverage levels obtained from the two technological platforms showed significant differences due to variations in analytical sensitivity.